Methods and compositions for delivering interleukin-1 receptor antagonist

ABSTRACT

Methods and compositions generating and using an interleukin-1 receptor antagonist (IL-1ra)-rich solution. Methods for generating and isolating interleukin-1 receptor antagonist include incubating a liquid volume of white blood cells and platelets with polyacrylamide beads to produce interleukin-1 receptor antagonist. The interleukin-1 receptor antagonist is isolated from the polyacrylamide beads to obtain the solution rich in interleukin-1 receptor antagonist. Methods for treating a site of inflammation in a patient include administering to the site of inflammation the solution rich in interleukin-1 receptor antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/031,803 filed Feb. 27, 2008, U.S. Provisional Application No.61/116,940 filed Nov. 21, 2008, and U.S. Provisional Application No.61/155,048 filed Feb. 24, 2009. The entire disclosures of each of theabove applications are incorporated herein by reference.

INTRODUCTION

The present technology relates to compositions comprising interleukin-1receptor antagonist, and methods for generating, isolating, anddelivering such compositions.

Interleukin-1 (IL-1) includes a family of cytokines that can stimulatelymphocytes and macrophages, activate phagocytes, increase prostaglandinproduction, contribute to degeneration of bone joints, increase bonemarrow cell proliferation, and are involved in many chronic inflammatoryconditions. IL-1 can be generated by macrophages, monocytes, anddendritic cells, and can be part of the inflammatory response againstinfection.

The mode of action of IL-1 can be mediated by interleukin-1 receptorantagonist protein (IL-1ra; also known as “IRAP”). IL-1ra binds to thesame receptor on the cell surface as IL-1, and thus prevents IL-1 fromsending a signal to that cell. IL-1ra is secreted from white bloodcells, including monocytes, macrophages, neutrophils, polymorphonuclearcells (PMNs), and other cells, and can modulate a variety of IL-1related immune and inflammatory responses, as described by Arend W P,Malyak M, Guthridge C J, Gabay C (1998) “Interleukin-1 receptorantagonist: role in biology” Annu. Rev. Immunol. 16: 27-55. Productionof IL-1ra is stimulated by several substances including adherentimmunoglobulin G (IgG), other cytokines, and bacterial or viralcomponents. IL-1ra is an important natural anti-inflammatory protein inarthritis, colitis, and granulomatous pulmonary disease.

IL-1ra can be used in the treatment of rheumatoid arthritis, anautoimmune disease in which IL-1 plays a key role, reducing inflammationand cartilage degradation associated with the disease. For example,Kinret™ (anakinra) is a recombinant, non-glycosylated form of IL-1ra(Amgen Manufacturing, Ltd., Thousand Oaks, Calif.). Various recombinantinterleukin-1 inhibitors and methods of treatment are described in U.S.Pat. No. 6,599,873, Sommer et al., issued Jul. 29, 2003; U.S. Pat. No.5,075,222, Hannum et al., issued Dec. 24, 1991; and U.S. ApplicationPublication No. 2005/0197293, Mellis et al., published Sep. 8, 2005 Inaddition, methods for producing IL-1ra from body fluids, including theuse of autologous fluids, are described in U.S. Pat. No. 6,623,472,Reincke et al., issued Sep. 23, 2003; U.S. Pat. No. 6,713,246, Reineckeet al., issued Mar. 30, 2004; and U.S. Pat. No. 6,759,188, Reinecke etal., issued Jul. 6, 2004.

Compositions and methods using IL-1ra are known in the art. For example,IL-1ra has been delivered as part of a composition with hyaluronic acid,as described in U.S. Pat. No. 6,096,728, Collins et al., issued Aug. 1,2000. However, many such methods and compositions are associated withissues regarding stability and half-life of IL-1ra as well as the amountand rate of IL-1ra provided. Accordingly, improved methods of deliveringIL-1ra are desirable and would be useful in treating conditions andpathologies mediated by the interleukin-1 receptor, including themanagement of inflammation.

SUMMARY

The present technology provides methods for generating solutions rich ininterleukin-1 receptor antagonist and for administering such solutionsto the site of inflammation in a human or animal subject. Methods forgenerating such solutions include incubating a liquid volume of whiteblood cells and, optionally, platelets with polyacrylamide beads. Thebeads are then separated from the liquid volume, thereby isolating asolution rich in interleukin-1 receptor antagonist. The liquid volume ofwhite blood cells may be whole blood and/or platelet-rich plasma.

Methods of treating a condition mediated by the interleukin-1 receptorin a human or animal subject, such as inflammation, includeco-administering a solution rich in interleukin-1 receptor antagonistand fibrinogen. In various embodiments, such methods further compriseadministration of thrombin and calcium chloride to the subject. The siteof inflammation may be associated, for example, with arthritis, e.g.,osteoarthritis. Preferably, the solution of IL-1ra is autologous.

BRIEF DESCRIPTION OF THE DRAWINGS

The present technology will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 is a diagrammatic illustration of a first method to produce asolution of IL-1ra according to an embodiment of the present technology;

FIG. 2 is a partial cross-sectional view of a representative device usedfor isolating a liquid volume of white blood cells and plateletsaccording to one embodiment of the present technology;

FIGS. 3A and 3B are cross-sectional views of a representative device forincubating a volume of white blood cells and platelets withpolyacrylamide beads according to one embodiment of the presenttechnology;

FIG. 4 is a diagrammatic illustration of a second method to produce asolution of IL-1ra according to an embodiment of the present technology;

FIG. 5 is a diagrammatic illustration of a third method to produce asolution of IL-1ra according to an embodiment of the present technology;

FIG. 6 is a diagrammatic illustration of a fourth method to produce asolution of IL-1ra according to an embodiment of the present technology;

FIG. 7 is blood component isolation device which may be used in methodsof the present technology;

FIG. 8 is a side view of the blood component isolation device of FIG. 7,illustrating an interior portion of a main chamber of the device;

FIG. 9 is a diagrammatic illustration of a method for delivering IL-1raaccording to an embodiment of the present technology; and

FIG. 10 is a partial cross-sectional view of a representative device fordelivering IL-1ra according to one embodiment of the present technology.

It should be noted that the figures set forth herein are intended toexemplify the general characteristics of materials and methods amongthose of the present technology, for the purpose of the description ofcertain embodiments. These figures may not precisely reflect thecharacteristics of any given embodiment, and are not necessarilyintended to define or limit specific embodiments within the scope ofthis technology.

DESCRIPTION

The description of the following technology is merely exemplary innature of the subject matter, manufacture, and use of the technologydisclosed herein, and is not intended to limit the scope, application,or uses of any specific invention claimed in this application, or insuch other applications as may be filed claiming priority to thisapplication, or patents issuing therefrom.

FIG. 1 depicts a first method 100 for generating a solution rich inIL-1ra. Blood is drawn from a human subject at step 110. As discussedbelow, this blood may be used directly in step 130, or may be processedto create a blood fraction in step 120.

For example, as shown in step 120, the blood can be centrifuged toisolate platelet-rich plasma (PRP) containing white blood cells andplatelets, which may be located in the buffy coat layer followingsedimentation. One example of a device that may be used for isolatingplatelet-rich plasma at step 120 is shown in FIG. 2. In this regard, thedevice 200 includes a container 205, such as a tube, that is placed in acentrifuge after being filled with blood. The container 205 includes abuoy system having an isolator 210 and a buoy 215. The buoy 215 has aselected density which is tuned to reach a selected equilibrium positionupon centrifugation; this position lies between a more dense bloodfraction and a less dense blood fraction. During centrifugation, thebuoy 215 separates the blood within the container 205 into at least twofractions, without substantially commingling the fractions, bysedimenting to a position between the two fractions. In this regard, theisolator 210 and the buoy 215 define a layer comprising platelet-richplasma 220, while less dense platelet-poor plasma 225 generallyfractionates above the isolator 210, and more dense red blood cells 230generally fractionate below the buoy 215. Following centrifugation, asyringe or tube may then be interconnected with a portion of the buoysystem to extract the platelet-rich plasma, containing white bloodcells. In various embodiments, such devices may be used to generateplatelet-rich plasma that includes a platelet concentration up to about8-fold higher than whole blood and a white blood cell concentration upto about 5-fold higher than whole blood. The platelet rich plasma maycomprise from about 80% to about 90% of the white blood cells present inthe whole blood. Such devices that are commercially available includethe GPS® II Platelet Concentrate System, from Biomet Biologics, LLC(Warsaw, Ind., USA) and GPS® III Platelet Separation System, from BiometBiologics, LLC (Warsaw, Ind., USA).

Devices that may be used to isolate platelet-rich plasma at step 120 arealso described, for example, in U.S. Pat. No. 6,398,972, Blasetti etal., issued Jun. 4, 2002; U.S. Pat. No. 6,649,072, Brandt et al., issuedNov. 18, 2003; U.S. Pat. No. 6,790,371, Dolocek, issued Sep. 14, 2004;U.S. Pat. No. 7,011,852, Sukavaneshvar et al., issued Mar. 14, 2006;U.S. Application Publication No. 2004/0251217, Leach et al., publishedDec. 16, 2004 (incorporated by reference herein); U.S. ApplicationPublication No. 2005/0109716, Leach et al., published May 26, 2005(incorporated by reference herein); U.S. Application Publication No.2005/0196874, Dorian et al., published Sep. 8, 2005 (incorporated byreference herein); and U.S. Application Publication No. 2006/0175242,Dorian et al., published Aug. 10, 2006 (incorporated by referenceherein).

Other methods may be used to isolate platelet-rich plasma in step 120.For example, whole blood can be centrifuged without using a buoy system,whole blood may be centrifuged in multiple stages, continuous-flowcentrifugation can be used, and filtration can also be used. Inaddition, a blood component including platelet-rich plasma can beproduced by separating plasma from red blood cells using a slow speedcentrifugation step to prevent pelleting of the platelets. In otherembodiments, the buffy coat fraction formed from centrifuged blood canbe separated from remaining plasma and resuspended to form platelet-richplasma.

In addition to the GPS® Platelet Concentrate and Separation Systems, avariety of other commercially available devices may be used to isolateplatelet-rich plasma at step 120, including the Magellan™ AutologousPlatelet Separator System, commercially available from Medtronic, Inc.(Minneapolis, Minn., USA); SmartPReP™, commercially available fromHarvest Technologies Corporation (Plymouth, Mass., USA); DePuy (Warsaw,Ind., USA); the AutoloGel™ Process, commercially available fromCytomedix, Inc. (Rockville, Md., USA); the GenesisCS System,commercially available from EmCyte Corporation (Fort Myers, Fla., USA);and the PCCS System, commercially available from Biomet 3i, Inc. (PalmBeach Gardens, Fla., USA).

Referring again to FIG. 1, the blood drawn from the subject at step 110may be mixed with an anticoagulant prior to subsequent use in steps 120or 130. Suitable anticoagulants include heparin, citrate phosphatedextrose (CPD), ethylenediaminetetraacetic acid (EDTA), anticoagulantcitrate dextrose solution (ACD), and mixtures thereof. The anticoagulantmay be placed in the syringe used for drawing blood from the subject, ormay be mixed with the blood after it is drawn.

As shown at step 130 of FIG. 1, the platelet-rich plasma containingwhite blood cells and platelets from step 120 is contacted withpolyacrylamide beads. In some embodiments, the platelet-rich plasma isincubated with the polyacrylamide beads for a time effective to remove aportion of the liquid in the liquid volume of white blood cells andplatelets. The incubation may be carried out over a period from about 30seconds to about 72 hours and may be carried out at a temperature fromabout 20° C. to about 41° C. For example, the incubation may be fromabout one minute to about 48 hours, from about 5 minutes to about 12hours, or from about 10 minutes to about 6 hours. In some embodiments,the incubation is conducted at about 37° C. In some embodiments theplatelet rich plasma is not incubated, but is contacted with thepolyacrylamide beads for only so long as necessary to perform subsequentprocessing. The contacting may occur at ambient conditions, e.g., at atemperature of about 20-25° C.

Polyacrylamide beads used in step 130 can be formed by polymerizingacrylamide monomer using controlled and standardized protocols as knownin the art to produce relatively uniform beads formed of polyacrylamidegel. In general, polyacrylamide is formed by polymerizing acrylamidewith a suitable bifunctional crosslinking agent, most commonlyN,N′-methylenebisacrylamide (bisacrylamide). Gel polymerization isusually initiated with ammonium persulfate and the reaction rate isaccelerated by the addition of a catalyst, such asN,N,N′,N′-tetramethylethylenediamine (TEMED). In various embodiments,polyacrylamide beads comprise 0.5 micromole of carboxyl groups permilliliter of beads, imparting a slight anionic character (negativecharge). The beads are also typically resistant to changes in pH, andare stable in many aqueous and organic solutions. By adjusting the totalacrylamide concentration, the polyacrylamide gel can be formed in a widerange of pore sizes. Moreover, the polyacrylamide beads can be formed inmany sizes and can have relatively uniform size distributions. Bead sizemay range from several micrometers in diameter to several millimeters indiameter. For example, various types of Bio-Gel™ P polyacrylamide gelbeads (Bio-Rad Laboratories, Hercules, Calif., USA) have particle sizesranging from less than about 45 μm up to about 180 μm. Polyacrylamidebeads are also available from SNF Floerger (Riceboro, Ga., USA), PierceBiotechnology, Inc. (Rockford, Ill., USA), and Polymers, Inc.(Fayetteville, Ark., USA).

Once polymerized, polyacrylamide beads can be dried and stored in apowder-like form. The dry beads are insoluble in water but can swellconsiderably upon being rehydrated. Rehydration returns thepolyacrylamide beads to a gel consistency that can be from about two toabout three times the dry state size. Thus, dry polyacrylamide beads maybe used to absorb a portion of a liquid volume, including solutessmaller than the bead pore size, and can serve to concentrate the IL-1raproduced by the white blood cells. For example, combining drypolyacrylamide beads with the blood and/or platelet-rich plasma in step130 activates production of IL-1ra by the white blood cells and alsoreduces the total liquid volume as the dry beads rehydrate and swell.

Alternatively, or in addition, blood from step 110 that is not subjectedto centrifugation in step 120 can be combined with polyacrylamide beadsin step 130 and incubated. This option is illustrated in FIG. 1 by thearrow running directly from step 110 to step 130. In this case, thepolyacrylamide beads activate production of IL-1ra in the blood, but theconcentration of IL-1ra may be lower compared to using platelet-richplasma containing white blood cells or platelets or another liquidvolume of white blood cells where the cells have been concentratedrelative to whole blood.

White blood cells for use in step 130 may also be prepared using othermethods known in the art. For example, white blood cells may be preparedfrom whole blood by lysing red blood cells or by centrifugation of wholeblood utilizing a density gradient where the white blood cells sedimentto the bottom of a centrifuge tube. An example of density centrifugationincludes the Ficoll-Paque™ Plus (GE Healthcare Bio-Sciences, Piscataway,N.J., USA). In some cases, a density gradient may be used to furtherseparate mononuclear and polymorphonuclear cells. White blood cells mayalso be prepared from whole blood using filtration; an example includesthe Acelere™ MNC Harvest System (Pall Life Sciences, Ann Arbor, Mich.,USA).

Without limiting the mechanism, utility or function of the presenttechnology, the polyacrylamide beads may serve as an activator of IL-1raproduction by the white blood cells. Therefore, in the case of drypolyacrylamide beads, not only is liquid being absorbed from the volumeof white blood cells, thereby concentrating the IL-1ra formed, but thebeads further serve as a surface to stimulate IL-1ra production by thewhite blood cells. For example, IL-1ra collected using platelet-richplasma (containing white blood cells) obtained using a device accordingto FIG. 2, such as the GPS® II system, may yield about a 5-fold increasein IL-1ra concentration versus whole blood. The concentration of IL-1ramay then be increased about 40-fold or more to a final concentrationincrease of about 200-fold upon incubation and isolation of theIL-1ra-rich solution using a device according to FIG. 3, such as aPlasmax™ device, as described further below. Thus, the increase in theamount of IL-1ra may not be due to simply increasing the concentrationby reducing the volume of the sample, but may also be due to activationof the white blood cells and other growth factors from platelets by thepolyacrylamide beads to increase production and/or release of IL-1raduring the incubation.

Referring again to FIG. 1, following incubation with the polyacrymidebeads, an IL-1ra-rich solution is isolated from the beads, as indicatedat step 140. Isolation may be accomplished by drawing off the liquidvolume and leaving the beads. In some cases, the beads may be sedimentedby centrifugation prior to drawing off the IL-1ra-rich solution.Isolation may also be performed by filtration, where the polyacrylamidebeads are retained by a filter and the IL-1ra-rich solution passesthrough the filter using centrifugal force or by using vacuum, forexample. If the incubation with polyacrylamide beads at step 130utilizes dry polyacrylamide beads, the liquid volume may be reduced asthe beads swell upon rehydration, thereby concentrating the resultingIL-1ra-rich solution. To maintain the increased concentration, careshould be taken in the isolation step 140 so as to avoid compressing thebeads or drawing liquid out from the swollen beads. For example, highcentrifugal force or high vacuum may collapse the beads and/or drawliquid out of the internal volume of the beads.

In some cases, the incubation with polyacrylamide beads, as per step130, and the isolation of the resulting IL-1ra-rich solution, as perstep 140, may be performed using a single device. An example of a devicefor incubating white blood cells and platelets with polyacrylamide beadsis shown in FIGS. 3A and 3B. In this regard, the device 300 has an upperchamber 305 and a lower chamber 310. The upper chamber 305 has an endwall 315 through which the agitator stem 320 of a gel bead agitator 325extends. The device 300 also has an inlet port 330 that extends throughthe end wall 315 and into the upper chamber 305. The device 300 alsoincludes an outlet port 335 that communicates with a plasma concentrateconduit 340. The floor of upper chamber 305 includes a filter 345, theupper surface of which supports desiccated concentrating polyacrylamidebeads 350.

During use, a fluid 355 containing white blood cells and platelets isinjected to the upper chamber 305 via the inlet port 330 and mixed withthe polyacrylamide beads 350. The fluid 355 and polyacrylamide beads 350may be mixed by rotating the agitator stem 320 and the gel bead agitator325, to help mix the fluid 355 and beads 350. The mixed fluid 355 andpolyacrylamide beads 350 are then incubated for the desired time at thedesired temperature. The device 300 is then centrifuged so that liquidpasses to the lower chamber 310 while the polyacrylamide beads 350 areretained by a filter 345, thereby separating the polyacrylamide beads350 from the resulting solution 360 of IL-1ra that collects in the lowerchamber 310. The solution 360 may be removed from the device via outletport 335.

Exemplary devices of FIG. 3 are disclosed in U.S. ApplicationPublication 2006/0175268, Dorian et al., published Aug. 10, 2006; andU.S. Application Publication 2006/0243676, Swift et al., published Nov.2, 2006; both of which are incorporated by reference herein. Such adevice is commercially available as Plasmax™ Plus Plasma Concentrator,from Biomet Biologics, LLC (Warsaw, Ind., USA).

Referring again to FIG. 1, in step 150 the IL-1ra-rich solution isadministered to a human or animal subject (patient). The patientreceiving the IL-1ra-rich solution may be the same patient from whichthe blood in step 110 is derived. In this case, the method provides anautologous preparation of IL-1ra. Administration may be performed usingvarious means, such as by injection of the IL-1ra-rich solution using asyringe, surgical application, or application concomitant with anothersurgical procedure. It should be understood, however, that step 150 maycomprise any biomedically acceptable process or procedure by which theIL-1ra-rich solution is implanted, injected, or otherwise administeredin or in proximity to a site in order to mediate effects related tostimulation of the interleukin-1 receptor, such as inflammation. Forexample, for treating inflammation caused by arthritis, an autologousIL-1ra-rich solution may be administered to the patient via injection.Injection may be located at or into the synovial space of an inflamedjoint, or otherwise at or near the joint.

Referring to FIG. 4, a second method 400 for generating a solution richin IL-1ra is shown. In this case, blood is first drawn from a patient instep 410. Proceeding to step 420, the blood is centrifuged, to isolateplatelet-rich plasma. As with the method of FIG. 1, the platelet-richplasma may be isolated with a device according to FIG. 2, or any othersuitable device such as described regarding the method of FIG. 1. Inthis method, the dual buoy mechanism includes polyacrylamide beadsbetween the buoy 215 and isolator 210. The polyacrylamide beads may bedry or hydrated, as described in reference to step 130 for FIG. 1.

During centrifugation in step 420, platelet-rich plasma collects betweenthe buoy 215 and isolator 210 and comes in contact with thepolyacrylamide beads. The less dense platelet-poor plasma componentforms above the platelet-rich plasma and the denser red blood cellcomponent forms below. Once centrifugation is completed, the tubecontaining the separated blood components may be incubated for thedesired time and at the desired temperature, indicated by step 430. Inthis manner, IL-1ra is generated by the white blood cells within themixture of platelet-rich plasma and polyacrylamide beads located betweenthe buoy and isolator.

In cases where dry polyacrylamide beads are used, once centrifugation iscomplete in step 420, the upper platelet-poor plasma component and thelower red blood cell component may be removed from the tube prior toincubation, leaving the platelet-rich plasma and polyacrylamide beadmixture between the two buoy portions. Alternatively, the mixture ofplatelet-rich plasma and polyacrylamide beads may be removed from thetube. In either case, separation of the platelet-rich plasma andpolyacrylamide bead mixture from fluid contact with the platelet-poorplasma and the red blood cell component allows subsequent swelling andrehydrating of dry polyacrylamide beads to effectively reduce the liquidvolume of the platelet-rich plasma, further concentrating the resultingIL-1ra solution.

As shown at step 440, the IL-1ra-rich solution is isolated from thepolyacrylamide beads. Separation of the IL-1ra-rich solution from thebeads may be accomplished using various means, such as those describedin reference to step 140 in FIG. 1. As shown at step 450, theIL-1ra-rich solution is then administered to a patient. Administrationmay be performed using various means, such as those described inreference to step 150 in FIG. 1.

Referring to FIG. 5, a third method 500 for generating a solution richin IL-1ra is shown. Blood is drawn from the patient in step 510. A largevolume concentration device is used to filter the blood and effectivelyremove some of the blood components, as shown at step 520, in order toproduce platelet-rich plasma containing white blood cells and platelets.

A suitable device for use in step 520 includes a separator assembly anda concentrator assembly. The separator assembly captures red blood cellsin a filter, such as a felt filter. The filter has pores and passagewaysthat are sized to receive and entrap red blood cells duringcentrifugation. The device captures the red blood cells by rotatingblood at speeds in a balanced cylindrical separation chamber that islined with the filter, where the separation chamber and filter aresegmented by radially extending plates into separation zones. Therotational speed of the separation chamber allows separation ofplatelet-rich plasma, including white blood cells, in the separationzones.

The concentrator assembly can concentrate the platelet-rich plasma byabsorbing liquid in the platelet-rich plasma using dry polyacrylamidebeads, as described in reference to step 130 in FIG. 1. Theplatelet-rich plasma is contacted in a rotating concentrating chamberwith the polyacrylamide beads to produce a platelet-rich plasmaconcentrate while the beads are stirred. The platelet-rich plasma andpolyacrylamide bead mixture can then be incubated in the concentratorassembly to allow for the generation of IL-1ra, including any additionalconcentration of the solution due to swelling and absorption of liquidby the beads. The resulting IL-1ra-rich solution is collected byrotating the concentration chamber at a speed to separate platelet-richplasma concentrate from the beads. Such devices include the Vortech™Concentration System (Biomet Biologics, LLC, Warsaw, Ind., USA), and aredisclosed in U.S. Application Publication 2006/0175244, Dorian et al.,published Aug. 10, 2006 and U.S. Application Publication 2006/0175242,Dorian et al., published Aug. 10, 2006, which are hereby incorporated byreference. These devices may be used in lieu of or in addition to usingthe tube having a buoy described in reference to step 120 in FIG. 1 toprepare platelet-rich plasma including white blood cells and platelets.

As shown at step 530, the IL-1ra-rich solution is then administered to apatient. Administration may be performed using various means, such asthose described in reference to step 150 in FIG. 1.

Referring to FIG. 6, a fourth method 600 for generating a solution richin IL-1ra is shown. Blood is drawn from the patient, as shown at step610, and combined with polyacrylamide beads, as shown at step 620. Thepolyacrylamide beads may be dry or hydrated, as described in referenceto step 130 in FIG. 1. Filtration is then used in step 630 to separate avolume of white blood cells and the polyacrylamide beads from red bloodcells. Filtration may be accomplished using a single filter or a seriesof size exclusion filters to capture the white blood cells and thebeads, while other blood components, such as red blood cells, pass withone or more filtrates. Once the filtration is complete, the volume ofwhite blood cells and polyacrylamide beads is incubated, as shown atstep 640, in order to activate the production of IL-1ra and furtherreduce the liquid volume if dry polyacrylamide beads are used. Plateletsmay also be added to the volume of white blood cells during theincubation in step 640.

The IL-1ra-rich solution is isolated from the polyacrylamide beads instep 650. Various means of isolation may be used, such as by drawing offthe liquid volume and leaving the beads. In some cases, the beads aresedimented by centrifugation prior to drawing off the IL-1ra-richsolution. Isolation may also be performed by filtration, where thepolyacrylamide beads are retained by a filter and the IL-1ra-richsolution passes through the filter using force generated by a centrifugeor by using vacuum, for example. In some cases, the IL-1ra-rich solutionis isolated from the polyacrylamide beads by drawing the solutionthrough the same filter or series of filters used in step 630. TheIL-1ra-rich solution may be drawn into a fresh collection chamber, orinto a previously used filtrate collection chamber where the one or moreearlier filtrates have been removed. The IL-1ra-rich solution is thenadministered to the patient, as shown at step 660.

The various preparations of IL-ra-rich solutions produced by the presenttechnology may be sterilized by including a sterile filter to processthe final isolated IL-1ra product. Similarly, an antibiotic may beincluded in the polyacrylamide beads during incubation or added at oneor more of the various steps in the methods described herein.

The present technology provides improved methods for preparing solutionsrich in IL-1ra, including autologous IL-1ra-rich concentrated plasmasolutions, that reduce and/or substantially eliminate immunologicalissues that may arise when using non-autologous material or recombinantmaterial. In addition, since the IL-1ra is produced by the patient'scells, natural post-translational modifications, such as glycosylation,are already present. This is not the case with most recombinant proteinssince they are produced in prokaryotic hosts.

Solutions, e.g., concentrated plasma solutions, rich in IL-1ra of thepresent technology can be characterized as comprising viable whole bloodcells, and having increased concentrations of IL-1ra, serum tumornecrosis factor R1 (sTNF-r1), plasma proteins, and growth factorsrelative to whole blood. It is understood, however, the concentrationspresent in any given solution may vary depending on the initial levelsof components present in the whole blood or plasma used in the presentmethods, and that increases in concentration are relative to thoseinitial levels. In general, IL-1ra is present in the solutions atconcentrations of at least about 10,000 pg/ml, at least about 25,000pg/ml, or at least about 30,000 pg/ml. Plasma protein levels aretypically present at concentrations of at least about 50 mg/ml, at leastabout 80 mg/ml, at least about 100 mg/ml, at least about 200 mg/ml, orat least about 250 mg/ml. In particular, albumin is present at aconcentration of about 40 mg/ml, or at least about 100 mg/ml; andfibrinogen is present at a concentration of at least about 2 mg/ml or atleast about 4 mg/ml. sTNF-r1 is typically present at concentrationsgreater than whole blood (about 960 pg/ml), such as at least about 1000pg/ml, or greater than 1500 pg/ml, or greater than about 2500 pg/ml.Increased concentrations of growth factors include: platelet-derivedgrowth factor PGDF-AB, at concentrations of greater than 50,000 pg/ml,or greater than 70,000 pg/ml; transforming growth factor TGF-β1, atconcentrations greater than 150,000 pg/ml, or greater than 190,000pg/ml; insulin-like growth factor IGF-1, at concentrations greater thanabout 140,000 pg/ml, or greater than 160,000 pg/ml; basic fibroblastgrowth factor bFGF, at concentrations greater than 150,000 pg/ml, orgreater than 170,000 pg/ml; and vascular endothelial growth factor VEGF,at concentrations greater than 1,200 pg/ml, or greater than 1,400 pg/ml.Concentrations of inflammatory cytokines (e.g., interleukin 1α,interleukin 1β, tumor necrosis factor-α and interleukin 10) aregenerally not significantly greater than whole blood, and may be lower.Exemplary specific levels of components are set forth in Table 1, below.

TABLE 1 Exemplary Composition Components Component Concentration plasmaproteins - total 286 mg/ml albumin 109 mg/ml fibrinogen 4.9 mg/ml IL-1ra34,000-108,000 pg/ml (whole blood = 200-800 pg/ml) sTNF-RI 270-3,450pg/ml (whole blood = 960 pg/ml) IL-1α below detection limit IL-1β 22pg/ml (whole blood = below detection limit) TNF-α below detection limitIL-10 1.6-9.06 pg/ml (whole blood = 4.53 pg/ml) Growth factors PDGF-AB73,201 pg/ml TGF-β1 194,076 pg/ml IGF-1 160,000 pg/ml bFGF 176 pg/mlVEGF 1,464 pg/ml

The IL-1ra-rich solutions may be administered to mediate effects of IL-1and attenuate signaling via the interleukin-1 receptor. The IL-1ra-richsolution may be used to block the biologic activity of naturallyoccurring IL-1, including inflammation and cartilage degradationassociated with arthritis, by competitively inhibiting the binding ofIL-1 to the interleukin-1 type receptor, which is expressed in manytissues and organs. For example, bone resorption and tissue damage suchas cartilage degradation as a result of loss of proteoglycans due toIL-1 may be treated by administration of the IL-1ra-rich solution. Inpatients with arthritis, endogenous IL-1ra may not be found in effectiveconcentrations in synovium and synovial fluid to counteract IL-1concentrations in these patients, and hence the present IL-1ra-richsolution may be administered to treat these conditions and these sites.Dosing, administration, and frequency of treatment may be modified basedon established medical practices to achieve effective treatment.

The present technology further provides methods for delivering IL-1ra.Such delivery methods provide a solution of IL-1ra and fibrinogen wherethe fibrinogen is activated to form a fibrin matrix that protects andretains the IL-1ra at a treatment site. The fibrin matrix can be formedin situ upon delivery of the IL-1ra.

Fibrinogen can be cross-linked into a three-dimensional matrix byactivation with a clotting agent and calcium. Suitable clotting agentsinclude thrombin (e.g., bovine, recombinant human, pooled human, orautologous), autologous clotting protein, and polyethylene glycol.Calcium may be in the form of a calcium salt, such as calcium chloride.

In some embodiments, the clotting agent comprises an autologous clottingprotein, as a clotting fraction or composition derived from a bloodobtained from the subject to be treated. A suitable clotting fractioncan be obtained by a process of: loading whole blood or plasma with acalcium solution (e.g., calcium chloride in ethanol) into a bloodisolation device; heating the whole blood or plasma for at least about20 minutes, at a temperature of at least about 20° C.; and isolating theclotting fraction. The isolating may be performed by centrifuging theheated whole blood or plasma. A suitable isolation device is depicted inFIGS. 7 and 8. Such a device is commercially available as the Clotalyst™Autologous Thrombin Collection System, sold by Biomet Biologics LLC,Warsaw, Ind., USA.

With reference to FIGS. 7 and 8, the blood separation device 700generally includes a body having a cylindrical wall along with a firstend 704 and a second end 706 that define a main chamber 702. At thefirst end 704 is a first port 708, a second port 710, a third port 712,a vent 713, and a filter 714. Each of the first port 708, the secondport 710, the third port 712, and the vent 713 extend through the firstend 704 and permit fluid communication between an exterior of the device700 and the main chamber 702. The first port 708 can be covered with afirst cap 716, the second port 710 can be covered with a second cap 718,and the third port 712 can be covered with a third cap 720. A firstreplacement cap 722 for the first port 708 can be attached to the firstport 708 with a first tether 724. A first cover 726 can be secured tothe first replacement cap 722 when the first replacement cap 722 is notin use. A second replacement cap 728 for the second port 710 can beattached to the second port 710 with a second tether 730. A second cover732 can be secured to the second replacement cap 728 when the secondreplacement cap 128 is not in use.

The first port 708 and the second port 710 each include a stop valve toprevent materials, such as glass beads 740, from exiting the mainchamber 702 through the first and the second ports 708 and 710. Thevalves can be any suitable valve, such as a duck-billed valve.

With particular reference to FIG. 8, the third port 712 includes anelongated tube portion 734 that extends within the main chamber 702. Theelongated portion 734 extends from the first end 704 to a depth withinthe main chamber 702 to permit withdrawal of select materials, such asthrombin and other blood clotting factors, from within the main chamber702. For example and as further described below, where the main chamber702 includes whole blood, reagents (e.g., a calcium solution comprisingcalcium compound dissolved in ethanol or other suitable solvent),anticoagulant, and glass beads, incubation and centrifugation of thismixture forms a clotted mass of about including red blood cells, bloodplasma, and glass beads at the second end 706 of the main chamber 702.On top of the clotted mass, at the side of the clotted mass nearest thefirst end 704, an effluent is formed comprising thrombin and variousother clotting factors. The clotted mass at the second end 706 can bevisually distinguished from the effluent. In order to extract thrombinand the other clotting factors using the elongated tube portion 734, theelongated tube portion 734 extends to a depth within the main chamber702 that is approximately level with the portion of the effluent closestto the clotted mass.

A tip 736 is provided at a distal end of the elongated portion 734. Thetip 736 extends from the elongated portion 734 at about a right angle.The tip includes a recess or notch 737. Two support posts 739 extendradially from the elongated portion 734 approximately at the tip 736 tocontact an interior of the main chamber 702. The support posts 739 biasthe tip 736 against the interior of the main chamber 702 to retain thetip 736 at a constant position in the main chamber 702. While the tip736 contacts the interior of the main chamber 702, the notch 737provides an opening or clearance between the interior wall of the mainchamber 702 and the tip 736 to permit the passage of material throughthe notch 737 and into the tip 736. The tip 736 helps to maximize theamount of materials withdrawn through the elongated portion 734,particularly when the main chamber 702 is tilted to bring additionalmaterials surrounding the tip 736 to the notch 737. The two supportposts 739 and the tip 736 help center the elongated portion 734 in themain chamber 702.

The ports 708, 710, and 712 are sized to cooperate with a suitable fluiddelivery or transport device, such as a syringe. For example, the firstport 708 can be sized to cooperate with a reagent syringe to permitpassage of reagent through the first port 708 and into the main chamber702; the second port 710 can be sized to cooperate with a blood syringeto permit passage of blood through the second port 710 and into the mainchamber 702; and the third port 712 can be sized to cooperate with asyringe to permit withdrawal of blood components, such as thrombin andother clotting factors, from within the main chamber 702.

The filter 714 can be any suitable filter for filtering materials asthey are withdrawn from within the main chamber 702 through the thirdport 712. The filter 714 includes a polyester screen that is mountedatop the first port 708 and the second port 710. The polyester screenincludes openings that are in the range of about 15 microns to about 25microns in size. For example, the openings can be about 17 microns insize. In place of or in addition to, the filter 714, a filter similar tothe filter 714 can be provided in the elongated portion 734 or at thetip 736.

The main chamber 702 further includes an activator, such as glass beads740. The negatively charged surface of the glass beads activatesclotting and the release of blood clotting factors, which form theclotted mass at the second end 706 of the main chamber 702. The glassbeads 740 can be any suitable type of glass beads, such as boro-silicatebeads.

An exemplary procedure for producing a clotting agent using the deviceof FIG. 7 begins injection of a reagent comprising calcium chloride andethanol into the main chamber 702 through the first port 708. After thereagent has been injected, the first port 708 is closed using the firstreplacement cap 722. Blood with anticoagulant is injected into the mainchamber 702 through the second port 710. After the blood has beeninjected, the second port 710 is closed using the second replacement cap728. Optionally, the syringes and blood separation device 700 arepre-heated to a temperature of about 25° C.

The contents of the blood component separation device 700 are mixed byrepeatedly inverting the device 700, e.g. about twelve times, so as tocontact the blood with the glass beads. After mixing, the device isincubated The incubation process can be at a temperature and for aduration that will permit the contents of the device 700 to be heated atabout 25° C. for about 15 minutes. Upon completion of the incubationperiod, a clotted mass of red blood cells, blood plasma, and glass beadsforms at the second end 706 of the main chamber 702. After incubation iscomplete, the device 700 is shaken enough to dislodge and break-up anygel that may be present. The device 700 is then placed in a suitablecentrifuge and spun at about 3200 RPM's for about 15 minutes to separatethrombin from the remaining blood components. After centrifugation, aneffluent of thrombin and other clotting factors separates from theclotted mass. After centrifugation is complete, the third cap 720 isremoved and a suitable extraction device, such a syringe, is used toremove the effluent of thrombin and other clotting factors from withinthe main chamber 702 by way of the third port 712, the elongated portion734, and the tip 736.

Thus, the delivery method of the present technology may includeadministration of IL-1ra, fibrinogen, thrombin, and calcium to form afibrin matrix at the treatment site. Exogenous fibrinogen may be addedto a solution of IL-1ra, for example such as bovine thrombin, preferablyat 1000 U/mL. Or, the IL-1ra solution may already have an adequateamount of endogenous fibrinogen. In the case where the solution ofIL-1ra and/or fibrinogen or preparation thereof includes ananticoagulant, such as ACD-A (anticoagulant citrate dextrose solution),the addition of calcium (with thrombin) to activate the fibrinogenshould exceed the effective amount of any chelator in the anticoagulant.

The IL-1ra-rich solutions prepared using the present methods can providean increased concentration of endogenous fibrinogen relative to wholeblood. For example, output of the above methods employing polyacrylamidebeads and the device illustrated in FIG. 3 results in a solution rich inboth IL-1ra and fibrinogen relative to whole blood. Such a device iscommercially available as the Plasmaxm Plus Plasma Concentrator, fromBiomet Biologics, LLC (Warsaw, Ind., USA) and includes those devices andmethods of use described in U.S. Application Publication 2006/0175268,Dorian et al., published Aug. 10, 2006; and U.S. Application Publication2006/0243676, Swift et al., published Nov. 2, 2006; both of which areincorporated by reference herein. This IL-1ra-rich and fibrinogen-richsolution may be used to treat the subject from which the original wholeblood was derived; i.e., autologous treatment.

An IL-1ra-rich and fibrinogen-rich solution, prepared using the abovemethods using polyacrylamide beads with the Plasmax™ Plus PlasmaConcentrator, provides a solution having about a 3-fold (3×) increase infibrinogen concentration relative to whole blood. The fibrin matrix/clotformed from the 3× higher concentration of fibrinogen is moresubstantial than a fibrin clot made from baseline fibrinogen levels andis more resistant to breakdown and resorption.

Referring to FIG. 9, a diagrammatic illustration for delivering IL-1ra900 is shown. At step 910, a solution of IL-1ra (IL-1ra) is provided.The IL-1ra (IL-1ra) solution may be prepared using the methods describedin the present disclosure. Exogenous fibrinogen is added to the IL-1ra(IL-1ra) solution in step 920. The exogenous fibrinogen may be preparedfrom a different source than the IL-1ra (IL-1ra) solution, such as adifferent patient, or may be bovine in origin. Or, the exogenousfibrinogen may be prepared from different starting material than theIL-1ra (IL-1ra) solution, but still from the same source or patient. Forexample, the IL-1ra (IL-1ra) solution and the exogenous fibrinogen maybe prepared from different blood samples taken from the same patient.Alternatively, as shown in step 930, a solution that is enriched in bothIL-1ra (IL-1ra) and fibrinogen is prepared, for example, by usingpolyacrylamide beads and the Plasmax™ device, as described herein. Asolution of thrombin and calcium is provided in step 940 and isco-administered with the solution of IL-1ra (IL-1ra) to a treatmentsite. Thereafter, as shown in step 950, the fibrin in the combinedsolutions cross-links in situ, forming a matrix at the treatment sitethat serves to protect, retain, and slow release of the IL-1ra (IL-1ra).

Delivery of IL-1ra may include co-administering a first solution ofIL-1ra and fibrinogen and a second solution of thrombin and calcium to asubject. In such embodiments, the first solution and second solution arekept separate until administered so that the fibrinogen does not form afibrin matrix until after the solutions are mixed and injected into atreatment site. The solutions may be mixed just before delivery to thetreatment site or may be mixed at the treatment site.

Referring to FIG. 10, a dual syringe device 1000 may be employed in amedically appropriate procedure. The dual syringe device 1000 includes afirst barrel 1005 and a second barrel 1010, both of which are connectedto a mixing chamber 1015. A first plunger 1020 is inserted into thefirst barrel 1005 and a second plunger 1025 is inserted into the secondbarrel 1010. The first plunger 1020 and the second plunger 1025 areconnected by a member 1030. The mixing chamber 1015 connects to acannula 1035. The dual syringe device 1000 contains a first solution1040 of IL-1ra and fibrinogen in the first barrel 1005, and a secondsolution 1045 of thrombin and calcium in the second barrel 1010. Duringco-administration, member 1030 is pushed toward the mixing chamber 1015such that the contents of both the first barrel 1005 and the secondbarrel 1010 are pushed into the mixing chamber 1015. The mixed firstsolution 1040 and second solution 1045 travel through the cannula 1035and form a fibrin-matrix 1050 at the treatment site 1055 within apatient's joint 1060.

In the embodiment shown in FIG. 10, the patient's joint 1060 is a kneejoint that includes a femur 1065, a tibia 1070, a fibula 1075, a patella1080, and cartilage 1085. It should be understood, however, that thetreatment site 1055 may be in any joint of a human patient or animal,including shoulders, elbows, wrists, ankles, hips, and the spinalcolumn. In addition, the present methods may be used to treatinflammation in sites within other tissues, such as muscle and tendon.

In some embodiments, the dual syringe device 1000 is used to pierce softtissue of the patient's joint 1060 to administer the mixed firstsolution 1040 and second solution 1045. For example, the cannula 1035may be a hollow needle such as a hypodermic needle. Alternatively, anincision may be made in the patient's joint 1060 to allow entry of thecannula 1035 so that the dual syringe device 800 may enter the treatmentsite 1055.

In some embodiments, which are not shown, the dual syringe device 1000does not have a mixing chamber 1015 and instead includes two cannulas1035, one leading from each barrel to the treatment site 1055. In thiscase, the first solution 1040 and second solution 1045 travel throughthe separate cannulas 1035 and mix together at the treatment site 1055to form a fibrin-matrix 1050. In some embodiments, two separatesingle-barreled syringe devices are employed in place of a dual syringedevice.

The fibrin matrix formed in the present delivery methods can reside atthe treatment site without increasing inflammation. The IL-1 ra withinthe fibrin matrix is protected from enzymatic degradation and may bindto the fibrin matrix so that is it slowly released from the matrix overtime. The methods consequently can provide sustained delivery of IL-1raas compared to injection of IL-1ra without the fibrin-matrix carrier.

The following specific examples are provided for illustrative purposesof how to make and use the compositions and methods of this technologyand, unless explicitly stated otherwise, are not intended to be arepresentation that given embodiments of this technology have, or havenot, been made or tested.

Example 1—Stimulation of IL-1Ra Production from Platelet-Rich Plasma

An IL-1ra-rich solution is created as follows. Whole blood (70 mL)anticoagulated (10%) with ACD-A (Braintree, Mass., USA) is drawn from 5healthy volunteers. A portion (10 mL) is reserved for a whole bloodmeasurement. Platelet-rich plasma (PRP) (6 mL) is produced using theGPS® II System (Biomet Biologics, LLC, Warsaw, Ind., USA). Completeblood counts (CBC) are collected for the whole blood and PRP samplesfollowing a validated procedure, as described in Woodell-May J E,Ridderman D N, Swift M J, Higgins J. “Producing Accurate Platelet Countsfor Platelet Rich Plasma: Validation of a Hematology Analyzer andPreparation Techniques for Counting” J Craniofac Surg (2005) September16(5):749-56. Following the PRP production, 5 mL of the PRP is added toa modified plasma concentration device (Plasmax™, Biomet Biologics LLC,Warsaw, Ind., USA) and incubated with polyacrylamide desiccating beadsin the device for 24 hours at room temperature. Following incubation,the plasma concentration device is centrifuged to separate the serumfraction.

To analyze baseline IL-1ra levels at time zero, the whole blood and PRPsamples are activated with 50 μL of thrombin and 10% CaCl₂) (1,000units/mL). A blood clot is formed and incubated for 30 minutes at roomtemperature. Following incubation, the clot is centrifuged for 5 minutesat 3,000 rpm. Serum is collected from the clots and retained for ELISAanalysis. The serum fraction from the plasma concentrator does notrequire activation by thrombin, and is tested directly. All samples areanalyzed for IL-1ra using an ELISA kit (IL-1ra Quantikinem Kit, R&DSystems, Minneapolis, Minn., USA).

Illustrative data is presented as mean±standard deviation. Statisticalsignificance is evaluated with a Student's t-test (a=0.05). Acorrelation analysis is used to compare IL-1ra output and complete bloodcounts (CBC) data.

Illustrative results are as follows. IL-1ra release from whole blood,PRP, and the concentrated serum are compared, with the data shown inTable 2. The whole blood and PRP IL-1ra are collected from serumfollowing thrombin activation. The plasma concentration device is usedto produce blood serum without the addition of thrombin.

TABLE 2 IL-1ra values measured by ELISA from five human donors Serumfraction from Whole blood PRP plasma concentrator Donor (pg/mL) (pg/mL)(pg/mL) 1 317 1,233 40,825 2 153 600 36,944 3 183 569 20,967 4 324 74239,827 5 110 1,073 40,438 Mean: 217 ± 98 843 ± 295 35,800 ± 8,432 Foldincrease: 4.6X    195X

The PRP samples result in an 8.1-fold increase in platelets, 5.2-foldincrease in total white blood cells (WBCs), an 8.7-fold increase in themonocyte fraction of the WBCs, and a 2.6-fold increase in the PMNfraction of the WBCs, as shown in Table 3. The IL-1ra production in thewhole blood and PRP samples is correlated most closely to the WBCconcentration (R²=0.82). The 4.6-fold increase in the PRP is probablydue to the increase in WBCs, and both the whole blood and PRP IL-1ravalues can be considered baseline IL-1ra content. This is in contrast tothe 195-fold increase in IL-1ra following the 24-hour incubation in theplasma concentrator. This plasma concentration device typically resultsin a 3-fold increase in plasma protein concentration due to a volumereduction caused by the desiccation process. This 3-fold decrease involume does not account for the levels of increase seen in the amount ofIL-1ra. Therefore, this level of increase indicates stimulation of WBCsto produce IL-1ra during the 24-hour incubation period.

TABLE 3 Average CBC values for blood and PRP Platelets WBC Monocytes PMNSample (K/μL) (K/μL) (K/μL) (K/μL) Whole blood 200 ± 28  5.5 ± 1.5 0.4 ±0.008 3.1 ± 1.3 PRP 1630 ± 210 28.5 ± 4.8 3.8 ± 1.3  8.0 ± 2.4 Foldincrease  8.1X  5.2X  8.7X  2.6X R² 0.57 0.82 0.68 0.77

Increased levels of IL-1ra are detected in PRP samples. Furtherprocessing of the PRP in a plasma concentration device can result ineven greater increased levels in IL-1ra. The baseline serum values ofIL-1ra (217±98 pg/mL) are similar to results found in another study(73±4.8 pg/mL), described in Meijer H, Reinecke J, Becker C, Tholen G,Wehling P. “The production of anti-inflammatory cytokines in whole bloodby physico-chemical induction” Inflamm. Res. 2003 October; 52(10):404-7,even though significant variability between donors can exist. The IL-1raserum levels are statistically higher in the PRP and serum output of theplasma concentrator than in the baseline serum levels. The 24-hourincubation of the PRP in the plasma concentration device results in adose of IL-1ra (35,800±8,432 pg/mL) that is higher than the previouslyreported data from the 24-hour incubation in the ACS device (10,254±165pg/mL).

Correlation analysis demonstrates that IL-1ra production is more closelycorrelated with the increase in WBCs than the platelet content. TheIL-1ra levels do not correlate as closely with the monocytes populationin the PRP. This is not surprising since the monocytes are notactivated, and the serum is collected by thrombin activation of theplasma. However, it is probable that the monocytes, once activated inthe plasma concentration device, participate in the significantproduction of IL-1ra seen.

Example 2—Elution of IL-1Ra (IL-1Ra) from a Concentrated-Plasma Matrix

Anticoagulated blood (120 cc) is collected from 5 human donors.Platelet-rich plasma (PRP) is prepared using GPS®III disposables (BiometBiologics LLC, Warsaw, Ind., USA). PRP is loaded into modified plasmaconcentration devices (Plasmax®, Biomet Biologics LLC, Warsaw, Ind.,USA) and processed. The output is divided into 4 groups; IL-1ra inconcentrated plasma with and without thrombin activation (1000 U/ml in1MCaCl₂), or cell-free IL-1ra with and without thrombin activation.IL-1ra is measured using ELISA (R&D Systems) over time.

Unclotted APS produces an average of 47.1±2.1 ng over 24 hrs (p=0.34).The cell-free samples produce 33.7±1.5 ng without changing over 24 hrs(p=0.38). Once clotted, the elution of IL-1ra is slowed, with only 28%being eluted after 10 hours. Release in the cell-free samples is alsodelayed, but eluted 100% of available IL-1ra after 10 hours.

The examples and other embodiments described herein are exemplary andnot intended to be limiting in describing the full scope of compositionsand methods of this technology. Equivalent changes, modifications andvariations of specific embodiments, materials, compositions and methodsmay be made within the scope of the present technology, withsubstantially similar results.

1. A method for generating a solution rich in interleukin-1 receptorantagonist comprising: (a) contacting a liquid volume of white bloodcells with polyacrylamide beads; (b) separating the polyacrylamide beadsfrom the liquid volume to obtain a solution rich in interleukin-1receptor antagonist.
 2. The method for generating a solution rich ininterleukin-1 receptor antagonist according to claim 1, wherein theliquid volume of white blood cells is whole blood or platelet-richplasma.
 3. The method for generating a solution rich in interleukin-1receptor antagonist according to claim 1, wherein the contactingcomprises incubating the platelet-rich plasma with the polyacrylamidebeads for a time of from about 30 seconds to about 24 hours.
 4. Themethod for generating a solution rich in interleukin-1 receptorantagonist according to claim 2, wherein the contacting furthercomprises mixing whole blood with an anticoagulant.
 5. The method forgenerating a solution rich in interleukin-1 receptor antagonistaccording to claim 4, wherein the anticoagulant comprises anticoagulantcitrate dextrose solution.
 6. The method for generating a solution richin interleukin-1 receptor antagonist according to claim 2, wherein theliquid volume of white blood cells is prepared by centrifuging wholeblood to increase the concentration of white blood cells and plateletsrelative to whole blood.
 7. The method for generating a solution rich ininterleukin-1 receptor antagonist according to claim 6, wherein thecentrifuging whole blood comprises: (i) loading the whole blood into atube comprising a buoy disposed in the tube, wherein the buoy has adensity such that the buoy reaches an equilibrium position uponcentrifugation of whole blood in the tube, the position being between afraction comprising red blood cells and a fraction comprising whiteblood cells; (ii) centrifuging the tube so that the buoy defines aninterface between a fraction comprising red blood cells and a fractionthat comprises white blood cells; and (iii) collecting the fractioncomprising white blood cells. 8-11. (canceled)
 12. The method forgenerating a solution rich in interleukin-1 receptor antagonistaccording to claim 1, wherein the solution comprises interleukin-1receptor antagonist at a concentration of at least about 10,000 pg/ml.13. (canceled)
 14. The method for generating a solution rich ininterleukin-1 receptor antagonist according to claim 2, wherein thesolution comprises soluble Tumor Necrosis Factor Receptor 1 (sTNF-R1) ata concentration greater than the concentration of the sTNF-R1 present inthe whole blood or plasma.
 15. A solution rich in interleukin-1 receptorantagonist made according to process of claim 1, said solutioncomprising: (i) interleukin-1 receptor antagonist at a concentration ofat least about 30,000 pg/ml; (ii) soluble Tumor Necrosis Factor Receptor1 (sTNF-R1) at a concentration greater than the concentration of thesTNF-R1 present in the whole blood or plasma; (iii) viable white bloodcells; and (iv) growth factors. wherein the total concentration ofplasma proteins is at least about 80 mg/ml.
 16. A method of treatinginflammation in a patient comprising administering to the site ofinflammation a solution rich in interleukin-1 receptor antagonist madeaccording to the process of claim
 2. 17. The method of treatinginflammation in a patient according to claim 16, wherein theinflammation is associated with osteoarthritis.
 18. The method oftreating inflammation in a patient according to claim 16, furthercomprising administering fibrinogen, thrombin, and calcium to the siteof inflammation.
 19. The method of treating inflammation in a patientaccording to claim 18, comprising co-administering (i) a first solutioncomprising the interleukin-1 receptor antagonist and fibrinogen, and(ii) a second solution comprising thrombin and calcium. 20-30.(canceled)
 31. A solution rich in interleukin-1 receptor antagonistcomprising: (i) interleukin-1 receptor antagonist at a concentration ofat least about 25,000 pg/ml; (ii) soluble Tumor Necrosis Factor Receptor1 (sTNF-R1); (iii) viable white blood cells; and (iv) growth factors.wherein the total concentration of plasma proteins is at least about 80mg/ml.
 32. The solution rich in interleukin-1 receptor antagonistaccording to claim 31, wherein the sTNF-R1 is present at a concentrationgreater than about 1500 pg/ml.
 33. The solution rich in interleukin-1receptor antagonist according to claim 31, wherein the totalconcentration of plasma proteins is at least about 200 mg/ml.
 34. Thesolution rich in interleukin-1 receptor antagonist according to claim31, further comprising fibrinogen at a concentration of at least about 2mg/ml, and albumin at a concentration of at least about 40 mg/ml.
 35. Acomposition comprising: (a) the solution rich in interleukin-1 receptorantagonist according to claim 31 and (b) thrombin.
 36. The compositionaccording to claim 35, wherein the thrombin is present in a clottingcomposition made by a process comprising: (a) loading whole blood orplasma and a calcium solution into a blood isolation device; (b) heatingthe whole blood or plasma for at least about 20 minutes, at atemperature of at least about 20° C.; and (c) isolating athrombin-containing clotting composition by centrifuging the heatedwhole blood or plasma.